ZytoZytoZytoZytoDotDotDotDot 2C2C2C2C SPEC HER2SPEC HER2SPEC HER2SPEC HER2/CEN 17/CEN 17/CEN 17/CEN 17 Probe KitProbe KitProbe KitProbe Kit

C-3022-40 40

C-3022-10 10

For the detection of the human HER2 gene and alpha-satellites of chromosome 17 by chromogenic in situ

hybridization (CISH)

. .

. .

In vitro diagnostic medical device

according to EU directive 98/79/EC

As of: February 1, 2011 (5.0)

Trademarks:Trademarks:Trademarks:Trademarks:

ZytoVision® and ZytoDot ® are trademarks of ZytoVision GmbH.

ContentsContentsContentsContents

1. Scope of Application .................................................................... 1

2. Basic Principles ........................................................................... 1

3. Safety Precautions and Disposal ................................................... 1

4. The ZytoDot 2C SPEC HER2/CEN 17 Probe Kit .............................. 2

4.1 Components .......................................................................... 2

4.2 Storage and Shelf Life ............................................................. 3

4.3 Test Material .......................................................................... 3

4.4 Additional Materials ................................................................ 3

4.5 Important Information ............................................................. 4

5. The ZytoDot 2C SPEC HER2/CEN 17 Probe Kit Protocol ................. 4

5.1 Preparatory Steps ................................................................... 4

5.2 Pretreatment (Dewax/Proteolysis) [day 1] .................................. 5

5.3 Denaturation and Hybridization [day 1] .................................... 6

5.4 Post-Hybridization and Detection [day 2] .................................. 6

6. Interpretation of Results................................................................ 7

6.1 CISH Results ........................................................................... 7

6.2 Positive Control ...................................................................... 9

7. Literature .................................................................................. 10

8. Problems and Possible Causes ................................................... 11

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1.1.1.1. Scope of ApplicationScope of ApplicationScope of ApplicationScope of Application

The ZytoDot 2C SPEC HER2/CEN 17 Probe Kit is designed to be used for the detection of the human HER2 gene as well as chromosome 17 alpha-satellites in either formalin-fixed, paraffin-embedded tissue or cell samples by chromo-genic in situ hybridization (CISH).

Interpretation of results must be made within the context of the patient’s clinical history with respect to further clinical and pathologic data of patient by a quali-fied pathologist.

2.2.2.2. Basic PrinciplesBasic PrinciplesBasic PrinciplesBasic Principles

The presence of certain nucleic acid sequences in cells or tissue can be detected with in situ hybridization using labeled DNA probes. The hybridization results in duplex formation of sequences present in the test object and the specific gene probe.

The ZytoDot 2C SPEC HER2/CEN 17 Probe Kit uses the ZytoDot 2C SPEC HER2/CEN 17 Probe EmaNOF. The probe contains digoxigenin-labeled polynu-cleotides, which target sequences of the HER2 gene and DNP-labeled polynu-cleotides, which target alpha-satellites of the centromere of chromosome 17.

Duplex formation of the labeled probe can be visualized using primary (un-marked) antibodies, which are detected by secondary polymerized enzyme-conjugated antibodies. The enzymatic reactions of the substrates lead to the formation of strong permanent red and green signals that can be visualized by light microscopy at a 40x dry lens.

3.3.3.3. Safety Precautions and DisposalSafety Precautions and DisposalSafety Precautions and DisposalSafety Precautions and Disposal

� Read the operating instructions prior to use!

� Do not use the reagents after the expiry date has been reached!

� Avoid any cross-contamination and micro-bacterial contamination of the reagents!

� Some of the system components contain substances (in low concentrations and volumes) that are harmful to health. Avoid any direct contact with the reagents. Take appropriate protective measures (use disposable gloves, protective glasses, and lab garments)!

� If reagents come into contact with skin, rinse skin immediately with copious quantities of water!

� Never pipet solutions with your mouth!

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� The disposal of reagents must be carried out in accordance with local regulations!

� A material safety data sheet is available on request for the professional user!

4.4.4.4. ThThThThe e e e ZytoZytoZytoZytoDotDotDotDot 2C 2C 2C 2C SPEC HER2SPEC HER2SPEC HER2SPEC HER2/CEN 17/CEN 17/CEN 17/CEN 17 Probe Probe Probe Probe KitKitKitKit

4.14.14.14.1 ComponentsComponentsComponentsComponents

The kit is made up of the following components:

CodeCodeCodeCode ComponentComponentComponentComponent QuantityQuantityQuantityQuantity

ContainerContainerContainerContainer 40404040 10101010

PT2 Heat Pretreatment Solution EDTA 500 ml 150 ml Screw-cap bottle (large)

ES1 Pepsin Solution 4 ml 1 ml Dropper bottle, white cap

PD12 ZytoDot 2C SPEC HER2/CEN 17 Probe

0.4 ml 0.1 ml Reaction vessel, brown lid

WB1 Wash Buffer SSC 500 ml 150 ml Screw-cap bottle (large)

WB5 20x Wash Buffer TBS 2x 50 ml 50 ml Screw-cap bottle

AB14 Anti-DIG/DNP-Mix 4 ml 1 ml Dropper bottle, yellow cap

AB13 HRP/AP-Polymer-Mix 4 ml 1 ml Dropper bottle, blue cap

SB6a AP-Red Solution A 0.4 ml 0.1 ml Dropper bottle, red cap (small)

SB6b AP-Red Solution B 15 ml 4 ml Dropper bottle, red cap

SB7a HRP-Green Solution A 0.8 ml 0.2 ml Dropper bottle, green cap (small)

SB7b HRP-Green Solution B 15 ml 4 ml Dropper bottle, green cap

CS2 Nuclear Blue Solution 20 ml 4 ml Screw-cap bottle, black

MT4 Mounting Solution (alcoholic) 4 ml 1 ml Glass bottle, brown

SC2 HER2 Control Slide 2 1 Plastic pack

AP-Red reaction vessel 2 1 Graduated cup, red lid

HRP-Green reaction vessel 2 1 Graduated cup, green lid

Instruction manual 1 1

CCCC----3022302230223022----40404040 (40(40(40(40 tests)tests)tests)tests): Components EmaNOF, EbpNF, E^_NQF, E^_NPF, Ep_S~JÄF, Ep_T~JÄF, E`pOF, and EjqQF are sufficient for 40 reactions. Components EmqOF and Et_NF are sufficient for 7 staining jars of 70 ml each. Component Et_RF is sufficient for 27 staining jars of 70 ml each.

CCCC----3022302230223022----10101010 (10(10(10(10 tests)tests)tests)tests): Components EmaNOF, EbpNF, E^_NQF, E^_NPF, Ep_S~JÄF, Ep_T~JÄF, E`pOF, and EjqQF are sufficient for 10 reactions. Components EmqOF and Et_NF are sufficient for 2 staining jars of 70 ml each. Component Et_RF is sufficient for 14 staining jars of 70 ml each.

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4.24.24.24.2 Storage and Shelf LifeStorage and Shelf LifeStorage and Shelf LifeStorage and Shelf Life

The components of the kit must be stored at 2…8°C. If these storage conditions are followed, the kit will function, without loss of performance, at least until the expiry date printed on the label.

4.34.34.34.3 Test MaterialTest MaterialTest MaterialTest Material

The ZytoDot 2C SPEC HER2/CEN 17 Probe Kit has been optimized for the use with formalin-fixed, paraffin-embedded tissue and cell samples. When test material is used that has been fixed or embedded in a different manner (e.g. methanol/glacial-acetic-acid-fixed cells or blood smears) the test protocol may need to be adapted by the user. Our specialists are available to support you whenever adjustments are necessary.

We recommend the following tissue preparation:

� Fixation in 10% neutrally buffered formalin for 24 h at RT

In order to achieve optimum and uniform fixation and paraffin embedding, the sample size should not exceed 0.5 cm3.

� Standard processing and paraffin embedding

Use premium quality paraffin. Infiltration and embedding should be carried out at temperatures lower than 65°C.

� Prepare 3-5 µm microtome sections

Draw up the sections onto silane-coated or adhesion slides (e.g. HistoBond ®) and fix for 2-16 h at 50-60°C.

A HER2 Control Slide Ep`OF may be used as control:

Control slides were pre-baked for 15 min at 58°C. If desired, tissue sample can be mounted next to the control cell lines. In either case, control slides have to be baked at 60°C for 2-16 h.

4.44.44.44.4 Additional MaterialsAdditional MaterialsAdditional MaterialsAdditional Materials

The following reagents, materials, and equipment are not included in the kit:

Reagents and materials

- Adhesive pistol, including hot adhesive, or rubber cement (Fixogum)

- Ethanol 100%, denatured

- Deionized or distilled water

- Xylene

- Hydrogen peroxide (H2O2) 30%

- Methanol 100%

Equipment

- Water bath (80°C, boiling)

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- Hot plate

- Hybridization oven (heating oven)

- Staining jars, 50-80 ml

- Humidity chamber

- Pipet (10 µl, 1000 µl)

- Coverslips (22 mm x 22 mm, 24 mm x 32 mm)

- Light microscope

4.54.54.54.5 Important InformationImportant InformationImportant InformationImportant Information

The following should be kept in mind:

� The tissue and cell sections must not be allowed to dry during the hybridi-zation and washing steps!

� The temperature for denaturating and washing, described in the protocol, should be used as a guide. Dependent upon the age and the fixation step of the sample material, an increase or decrease in temperature of the denaturing or wash steps can lead to better hybridization results!

5.5.5.5. ThThThThe e e e ZytoZytoZytoZytoDotDotDotDot 2C 2C 2C 2C SSSSPEC HER2PEC HER2PEC HER2PEC HER2/CEN/CEN/CEN/CEN 17171717 Probe KitProbe KitProbe KitProbe Kit ProtocolProtocolProtocolProtocol

5.15.15.15.1 Preparatory StepsPreparatory StepsPreparatory StepsPreparatory Steps

Day 1:

• Preparation of ethanol series (70%, 90%, and 100% ethanol solutions): Dilute 7, 9, and 10 parts of 100% ethanol with 3, 1, and 0 parts of deionized or distilled water, respectively. These solutions can be stored in suitable contain-ers and re-used.

• Heat Pretreatment Solution EDTA EmqOF: Heat in a covered staining jar stand-ing in a boiling water bath to at least 95°C.

• Pepsin Solution EbpNF: Bring to room temperature before use.

• Preparation of 3% H2O2: Dilute 1 part of 30% H2O2 with 9 parts of 100% methanol.

Day 2:

• Wash Buffer SSC Et_NF: Prepare two staining jars with Wash Buffer SSC, one at room temperature (RT), the other heated to 75°C (depending on the number of slides, the temperature should be increased by 1°C per slide for more than two slides, but not exceed 80°C).

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• Preparation of 1x Wash Buffer TBS: Dilute 1 part of 20x Wash Buffer TBS Et_RF in 19 parts deionized or distilled water. Diluted 1x Wash Buffer TBS lasts for one week when stored at 2-8°C.

• Anti-DIG/DNP-Mix E^_NQF, HRP/AP-Polymer-Mix E^_NPF, Nuclear Blue Solution E`pOF, Mounting Solution (alcoholic) EjqQF: Bring to room temperatu-re before use.

• Preparation of AP-Red Solution: Prior to immediate use, add one drop (30 µl) of AP-Red Solution A Ep_S~F in a graduated cup (e.g. AP-Red reaction vessel), fill up to 1 ml with AP-Red Solution B Ep_SÄF and mix well.

Do not expose to ststststrongrongrongrong direct light.

• Preparation of HRP-Green Solution: Prior to immediate use, add two drops (2x 20 µl) HRP-Green Solution A Ep_T~F in a graduated cup (e.g. HRP-Green reaction vessel), fill up to 1 ml with HRP-Green Solution B Ep_TÄF and mix well.

5.25.25.25.2 PretreatmPretreatmPretreatmPretreatment (Dewax/Proteolysis)ent (Dewax/Proteolysis)ent (Dewax/Proteolysis)ent (Dewax/Proteolysis) [day 1][day 1][day 1][day 1]

N. Incubate slides for 10 min at 70°C (e.g. on a hot plate)

OK= Incubate slides for 2x 5 min in xylene

PK= Incubate for 3x 3 min in 100% ethanol

Alternatively, dewaxing protocols routinely used in immunohistochemistry proce-dures, e.g. 2x 15 min xylene, 2x 5 min 100% ethanol, 2x 5 min 96% ethanol, 1x 5 min 70% ethanol, can be used.

QK= Incubate slides for 5 min in 3% H2O2

RK= Wash 2x 1 min in deionized or distilled water

SK= Place slides in the pre-heated Heat Pretreatment Solution EDTA EmqOF and

incubate for 15 min

TK= Transfer slides immediately to deionized or distilled water, wash 2x 2 min

and drain off or blot off the water

UK= Apply (dropwise) Pepsin Solution EbpNF to the tissue/cell section and incu-

bate for 5 min at RT in a humidity chamber

Depending on multiple factors, e.g. nature and duration of fixing, thickness of sections, and nature of tissue/cells, different incubation times may be required. As an incubation guideline, we recommend an incubation time of 3-10 min for tissue and cell samples. As a general rule, we recommend to ascertain the opti-mum time for proteolysis in pre-tests.

VK= Immerse slides in deionized or distilled water and drain off or blot off the

water

NMK= Dehydration: in 70%, 90%, and 100% ethanol, each for 1 min

Air dry sections.

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5.35.35.35.3 Denaturation and HybridizationDenaturation and HybridizationDenaturation and HybridizationDenaturation and Hybridization [day 1][day 1][day 1][day 1]

NK= Vortex the ZytoDot 2C SPEC HER2/CEN 17 Probe EmaNOF and pipette 10 µl

each onto individual samples

Distribute dropwise on the whole target area to avoid local concentration of probe. Alternatively, add probe to the center of a coverslip and place coverslip upside down on target area. A gentle warming of the probe, as well as using a pipette tip, which has been cut off to increase the size of the opening, can make the pipetting process easier.

OK= Avoiding trapped bubbles, cover the samples with a coverslip (22 mm x 22 mm). Seal the coverslip, e.g. with a layer of hot glue from an adhesive pistol or with rubber cement

PK= Denature the slides at 78-80°C for 5 min, e.g. on a hot plate

QK= Transfer the slides to a humidity chamber and hybridize overnight at 37°C

(e.g. in a hybridization oven)

It is essential that the tissue/cell samples do not dry out during the hybridization step.

5.45.45.45.4 PostPostPostPost----Hybridization and DetectionHybridization and DetectionHybridization and DetectionHybridization and Detection [day 2][day 2][day 2][day 2]

NK= Carefully remove the rubber cement or glue=

OK Remove the coverslip by submerging in Wash Buffer SSC Et_NF at RT for

5 min

PK= Wash 5 min in Wash Buffer SSC Et_NF at 75-80°C

The Wash Buffer SSC should be pre-heated. Increase temperature by 1°C per slide for more than 2 slides, bbbbut do not exceed 80°Cut do not exceed 80°Cut do not exceed 80°Cut do not exceed 80°C. Check with a thermometer if necessary.

QK Wash 2x 1 min in deionized or distilled water

RK Immerse slides in 1x Wash Buffer TBS (prepared using t_R) and drain off

or blot off the 1x Wash Buffer TBS

SK Apply Anti-DIG/DNP-Mix E^_NQF dropwise (3-4 drops per slide) to the

slides and incubate for 15 min at 37°C in a humidity chamber

TK Wash 3x 1 min in 1x Wash Buffer TBS (prepared using t_R) and drain off

or blot off the 1x Wash Buffer TBS

UK Apply HRP/AP-Polymer-Mix E^_NPF dropwise (3-4 drops per slide) to the

slides and incubate for 15 min at 37°C in a humidity chamber

VK During the incubation, prepare AP-Red Solution by adding one drop (30 µl) of AP-Red Solution A Ep_S~F in a graduated cup (e.g. AP-Red reaction vessel), fill up to 1 ml with AP-Red Solution B Ep_SÄF and mix well

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Do not expose to strongstrongstrongstrong direct light.

NMK Wash 3x 1 min in 1x Wash Buffer TBS (prepared using t_R)

NNK Apply AP-Red Solution dropwise (3-4 drops per slide) to the slides and incubate for 10 min at RT (protect from strong direct light). If required, the incu-bation time can be shortened or extended (7-15 min)

NOK During the incubation, prepare HRP-Green Solution by adding two drops (2x 20 µl) of HRP-Green Solution A Ep_T~F in a graduated cup (e.g. HRP-Green reaction vessel), fill up to 1 ml with HRP-Green Solution B Ep_TÄF and mix well

NPK Wash slides 2 min in deionized or distilled water and drain off or blot off

the water

NQK Apply HRP-Green Solution dropwise (3-4 drops per slide) to the slides and incubate for 10 min at RT (protect from strong direct light). If required, the incu-bation time can be shortened or extended (5-15 min)

NRK Wash 2 min in deionized or distilled water

NSK= Counterstain the tissue or cell samples for 2 min with Nuclear Blue Solution E`pOF

The counterstaining time depends on the nature of tissue/cell used and should therefore be optimized. Avoid dark counterstaining, because it may obscure posi-tive staining signals.

NTK= Transfer slides into a staining jar and wash 2 min in running tap water

NUK= Dehydration: 3x 30 s in 100% ethanol (use very pure ethanol)

NVK= Incubate 2x 30 s in xylene (use very pure xylene)

Do not prolong the incubation time as this might result in loss of signals!

OMK= Avoiding trapped bubbles, cover the samples immediately with a coverslip (22 mm x 22 mm; 24 mm x 32 mm) by using Mounting Solution (alcoholic) EjqQF and air dry the slides for approx. 30 min

ONK= Evaluation of the sample material is carried out by light microscopy

6.6.6.6. Interpretation of ResultsInterpretation of ResultsInterpretation of ResultsInterpretation of Results

6.16.16.16.1 CISH ResultCISH ResultCISH ResultCISH Resultssss

The CISH hybridization signal of one single copy of a HER2 gene appears as a dark green-colored distinct dot-shaped signal, the signal of one single copy of a chromosome 17 centromeric region appears as bright red-colored distinct dot-shaped signal, which can be clearly distinguished from the background counterstained with hematoxylin. Visualization of signals should be performed using at least a 40x objective resulting in easily visible signals.

Do not use contrast enhancing filter lenses as this might distort the signal color. To obtain signals in bright colors, open the aperture diaphragm. Be sure to

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focus up and down when evaluating a nucleus as red and green signals might be located on top of each other.

Prior to signal enumeration, the tissue/cell section should be scanned for any possible intratumoral heterogeneity using a 10x or 20x objective. In case of heterogeneity, an area representative for the amplification status has to be chosen. For signal enumeration, areas of necrosis, overlapping nuclei, and nuclei with weak signal intensity should be avoided.

In normal diploid nuclei without gene amplification, 2 green and 2 red dot-shaped signals with smooth, rounded edges will be visible per nucleus (see fig. 1111). Due to mitosis, additional signals may be visible even in a small percentage of non-neoplastic cells. Occasionally, nuclei with missing signals may be observed in paraffin-embedded tissue sections.

In case of low gene amplifications (see fig. 3333) or chromosome 17 aneusomy (see fig. 2222), green HER2 gene specific signals will be visible as multiple dots or small clusters. Small clusters are irregularly shaped signals comprising an area of up to 5 dots. As a reference, a single green dot of a normal cell of the same slide must be used. Additionally, red signals of the centromere 17 control will be visible.

In case of high gene amplifications, a large number of green dots or large clusters, comprising an area greater than 5 dots, will be visible in the nuclei (see fig. 4444). As a reference, a single green dot of a normal cell of the same slide must be used. Red signals might be overlaid and might not be visible any more.

....

= red signal = green signal

....

= red signal = green signal

1)1)1)1) Normal cells 2)2)2)2) Aneusomy of chromosome 17

....

= red signal = green signal

....

= red signal = green signal

3)3)3)3) Low gene amplification 4) 4) 4) 4) High gene amplification

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In order to judge the specificity of the hybridization signals and to confirm the correct performance of the method, any hybridization should be accompanied by controls. We recommend using at least one control sample, in which the chromosome 17 and HER2 gene copy number is known. The polynucleotides contained in the ZytoDot 2C SPEC HER2/CEN 17 Probe EmaNOF which recognize the alpha-satellite-sequences of the centromere of chromosome 17 function in themselves as an internal control that a successful hybridization has occurred, as well as proving the integrity of the cellular DNA. A negative or unspecific result can be caused by multiple factors. For troubleshooting, please refer to chapter 8.

6.26.26.26.2 Positive ControlPositive ControlPositive ControlPositive Control

The included HER2 Control Slides Ep`OF should be used for monitoring the cor-rect performance of the CISH experiment. For use as an on-slide-control simply mount tissue sample of interest next to the cell lines of the control slide before baking the slide.

The HER2 Control Slide Ep`OF contains four different cell lines (see below) affect-ted by different levels of HER2 amplification and one tissue (canine myocardial muscle). Cells have been embedded in red-colored paraffin, fixed in 10% neu-tral buffered formalin (24 h, pH 7.0), and mounted on positively charged slides in a thickness of 4 µm. Slides were pre-baked for 15 min at 58°C.

The HER2 Control Slide Ep`OF is designed as follows:

.

.

.

. 1:1:1:1: no HER2 amplification, 1-2 HER2 and 1-2 CEN 17 signals per nucleus 2:2:2:2: no HER2 signals, no CEN 17 signals 3:3:3:3: no HER2 amplification, 1-2 HER2 and 1-2 CEN 17 signals per nucleus 4:4:4:4: chromosome 17 aneusomy, 3-6 HER2 and 3-6 CEN 17 signals per nucleus 5:5:5:5: high level HER2 amplification, large HER2 cluster per nucleus

Prior to use of HER2 Control Slide Ep`OF, remove the label, then name the slide with a pencil, and, if desired, mount tissue sample of interest for on-slide-control, bake slide at 60°C for a minimum of 2 h up to 16 h, and proceed with chapter 5.2 Pretreatment (Dewax/Proteolysis) using a pepsin incubation time of 10 min.

1111

3333 4444 5555

2222

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7.7.7.7. LiteratureLiteratureLiteratureLiterature

Bhargava R, et al. (2005) Am J Clin Pathol 123123123123: 237-43.

Coussens L, et al. (1985) Science 230230230230: 1132-9.

Hauser-Kronberger C, Dandachi N (2004) J Mol Histol 35353535: 647-53.

Hopman AHN, et al. (1997) Histochem Cell Biol 108108108108: 291-8.

Isola J, Tanner M (2004) Methods Mol Med 97979797: 133-44.

Kounelis S, et al. (2005) Anticancer Res 25252525: 939-46.

Mayr D, et al. (2009) Histopathology 55555555: 716-23.

Speel EJ, et al. (1994) J Histochem Cytochem 42424242: 1299-307.

Tsukamoto T, et al. (1991) Int J Dev Biol 35353535: 25-32.

Wilkinson DG: In Situ Hybridization, A Practical Approach, Oxford University Press (1992) ISBN 0 19 963327 4.

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8.8.8.8. Problems aProblems aProblems aProblems and Possible Causesnd Possible Causesnd Possible Causesnd Possible Causes

Any deviation from the operating instructions can lead to inferior staining results or to no staining at all.

ProblemProblemProblemProblem Possible causePossible causePossible causePossible cause ActionActionActionAction

Poor tissue morphology

Overdigested tissue Shorten the pepsin incubation time

Weak signals or no signals

No target sequence in test material Use control slides

Cell or tissue sample has not been fixed properly

Optimization of fixation time; check quality of fixative and its compatibility with in situ-hybridization systems

Over- or underdigested tissue Optimization of proteolytic pretreatment time

Denaturation temperature not correct

Check temperature; adjust temperature if necessary

Hybridization temperature not correct

Stringency wash temperature not correct

Antibody incubation temperature not correct

Hybridization time too short Hybridize for at least 12 h; extend hybridization time if necessary

Incubation with chromogenic substrate too short

Extend incubation time

Chromogenic substrates were prepared too early

Prepare chromogenic substrates prior to immediate use

Counterstaining too dark Reduce counterstaining time

Insufficient preparation of chromogenic substrates

Instead of using one drop of AP-Red Solution A use 30 µl, and instead of using two drops of HRP-Green Solution A use 40 µl, and fill up to 1 ml with the respective Solution B

Weak red signals AP-Red Solution was exposed to strong direct light

Do not prepare AP-Red Solution or perform staining in direct strong light

Weak green signals Incubation times of any washing steps after staining with HRP-Green too long

Do not exceed given incubation times

Counterstaining time too long Reduce counterstaining time

Green signals fades or merges

An unsuitable mounting solution has been used

Use only the mounting solution provided with the kit or xylene-based mounting solutions free of any impurities; do not use coverslip tape

Sections were not dehydrated properly

Use fresh ethanol and xylene solutions; use only xylene of “pure” quality

Substrate reaction is too intensive Shorten substrate incubation time; do not heat substrate solution over 25°C; incubate at room temperature only

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Uneven / in some parts only very light staining

Incomplete dewaxing Use fresh solutions; check length of dewaxing times

Reagent volume too small Ensure that the reagent volume is large enough to cover the tissue area

Air bubbles caught before hybridization or during mounting

Avoid air bubbles

Strong background staining

Slides not thoroughly rinsed Use fresh wash buffers and deionized or distilled water where indicated

Sections dried out any time during or after hybridization

Avoid sections being dried out; use humidity chamber; seal coverslip properly

Washing temperature following hybridization too low

Check temperature; adjust temperature if necessary

Prolonged substrate incubation time Shorten substrate incubation time

Endogenous levamisole-resistant alkaline phosphatase

Additional blocking with Bouin’s Solution or 1M citric acid free acid for 1-10 min

Section floats off the slide

Unsuitable slide coating Use silane-coated or adhesion slides